Promoter sequence obtained from rice and methods of use

ABSTRACT

Methods are provided by which  Oryza sativa  plants and seeds thereof may be modified to express a coding region of interest using a promoter sequence operatively linked to the coding region. The promoter sequence is an isolated  Oryza sativa  antiquitin (OsAnt1) promoter sequence including SEQ ID NO: 1. The coding region of interest may encode a nitrogen utilization protein, suitably alanine aminotransferase. Methods to develop  Oryza sativa  plants that have increased biomass and seed yield are also presented. Furthermore,  Oryza sativa  plants may be produced that maintain a desired yield while reducing the need for high levels of nitrogen application.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/644,453, filed Dec. 21, 2006, which claims the benefit of U.S.Provisional App. No. 60/753,848, filed Dec. 23, 2005.

FIELD OF INVENTION

The present invention relates to a promoter sequence obtained from rice,to methods of using the promoter sequence, and to plants including thepromoter sequence.

BACKGROUND OF THE INVENTION

Crop plants have a fundamental dependence on inorganic nitrogenousfertilizers, principally in the form of nitrate (NO₃ ⁻) and ammonium(NH₄ ⁺). Each year, approximately 85 to 90 million metric tons (MMt) ofnitrogenous fertilizers are added to the soil worldwide. This amount isup from only 1.3 MMt in 1930 and from 10.2 MMt in 1960. It is predictedto increase to 240 MMt by the year 2050 (Tilman et al., 1999, Proc. Nat.Acad. Sci. USA. 96: 5995-6000). It is estimated that 50% to 70% of theapplied nitrogen is lost from the plant-soil system. Because NO₃ ⁻ issoluble and not retained by the soil matrix, excess NO₃ ⁻ may leach intothe water and may be depleted by microorganisms.

It is important to improve the nitrogen use efficiency (NUE) of cropplants for two reasons. First, the use of commercial fertilizersaccounts for one of the major costs associated with the production ofhigh yielding crops. Second, it would be an environmental benefit toreduce the levels of nitrogenous fertilizers that are lost into theecosystem. Environmental effects include the deterioration of soilquality, pollution and health hazards.

Alanine is one of the more common amino acids in plants. Alanine issynthesized by the enzyme alanine aminotransferase (AlaAT) from pyruvateand glutamate in a reversible reaction, as shown in FIG. 1. Alanine isan amino acid that is known to increase under other specificenvironmental conditions such as drought and anaerobic stress (Muenchand Good, 1994, Plant Mol. Biol. 24:417-427; Vanlerberge et al., 1993,Plant Physiol. 95:655-658). Alanine levels are known to increasesubstantially in root tissue under anaerobic stress. As an example,alanine levels in barley roots increase 20-fold after 24 hours ofanaerobic stress. The AlaAT gene is induced by light in broom millet andwhen plants are recovering from nitrogen stress (Son et al., 1992, Arch.Biochem. Biophys. 289: 262-266). Vanlerberge et al. (1993) have shownthat in nitrogen-starved anaerobic algae, the addition of nitrogen inthe form of ammonia resulted in 93% of an N₁₅ label being incorporateddirectly into alanine. Thus, alanine appears to be an important aminoacid in stress response in plants.

U.S. Pat. No. 6,084,153 discloses the induction of AlaAT in the roots ofcanola plants and a resulting nitrogen efficient phenotype.

WO 01/55433 teaches the use of Brassica turgor gene-26 (btg26). Theturgor gene-26-like proteins have recently been named antiquitins (Tanget al., 2002, FEBS Lett. 516(1-3):183-186). Brassica napus plants weretransformed with constructs containing the AlaAT gene in operativelinkage with the btg26 promoter. The transgenic plants were shown tohave elevated levels of AlaAT in the root tissue.

US2005/0044585 discloses the use of promoters LeAMT1, LeNRT1, GmNRT2,KDC1, PHT1, GOGAT, OsRAB5 and ALF5 to direct root specific expression ofa gene encoding a nitrogen utilization protein, for example, AlaAT.

Increasing NUE within rice is also desired within the art. In 2006,worldwide acreage devoted to growing rice was 151,730,000 hectares withnitrogen consumption estimated at 11,963 MMT. Thus, improving NUE inrice would not only decrease the cost of crop production but wouldreduce the harmful environmental effects of nitrogen fertilizersincluding the development of “dead zones” in the world's oceans thatresult from the death and decomposition of massive algae blooms fed byexcessive nutrient runoff.

SUMMARY OF THE INVENTION

Objectives of the present invention are to provide a promoter sequenceobtained from Oryza sativa (rice), methods for using the promotersequence, and plants including the promoter sequence.

In one embodiment, the present invention provides methods by which Oryzasativa may be modified to express a target gene or coding region ofinterest using a promoter sequence operatively linked to the codingregion. In another embodiment, the present invention also providesmethods of producing Oryza sativa plants that have increased biomass andseed yield. By increasing the biomass and seed yield, Oryza sativaplants are provided that have an environmental benefit in that they canmaintain a desired yield while reducing the need for high levels ofnitrogen application.

An isolated Oryza sativa antiquitin (OsAnt1) promoter sequence includingSEQ ID NO: 1 and active fragments thereof are disclosed.

Also provided is a genetic construct including an OsAnt1 promotersequence including SEQ ID NO: and active fragments thereof operativelylinked with a coding region of interest encoding a target protein. Thetarget protein may be a nitrogen utilization protein, for example,alanine aminotransferase (AlaAT).

A vector including a genetic construct with an OsAnt1 promoter sequenceincluding SEQ ID NO: 1 and active fragments thereof operatively linkedwith a coding region of interest encoding a target protein. The targetprotein may be a nitrogen utilization protein, for example, AlaAT.

A method for directing expression of a target gene in an Oryza sativaplant is described. The method may include contacting and introducinginto an Oryza sativa plant the target gene in operative linkage with anOsAnt1 promoter sequence including SEQ ID NO: 1 and active fragmentsthereof and expressing the target gene. The target gene may encode anitrogen utilization protein, for example, AlaAT. Furthermore,expression of the target gene may be targeted to a particular tissue,for example, to the root of the plant.

Also described is a method for increasing biomass of an Oryza sativaplant by contacting and introducing into an Oryza sativa plant a targetgene in operative linkage with an OsAnt1 promoter sequence including SEQID NO: 1 and active fragments thereof. The target gene may encode anitrogen utilization protein, for example, AlaAT. Furthermore,expression of the target gene may be targeted to a particular tissue,for example, to the root of the plant.

A method for increasing seed yield of an Oryza sativa plant is alsodescribed. The method my include contacting and introducing into anOryza sativa plant a target gene in operative linkage with an OsAnt1promoter sequence including SEQ ID NO: 1 and active fragments thereof.The target gene may encode a nitrogen utilization protein, for example,AlaAT. Furthermore, expression of the target gene may be targeted to aparticular tissue, for example, to the root of the plant.

Also described is a transformed Oryza sativa plant including a targetgene in operative linkage with an OsAnt1 promoter sequence including SEQID NO: 1 and active fragments thereof. The target gene may encode anitrogen utilization protein, for example, AlaAT.

Oryza sativa plant seed including a target gene in operative linkagewith an OsAnt1 promoter sequence including SEQ ID NO: 1 and activefragments thereof are described. The target gene may encode a nitrogenutilization protein, for example, AlaAT.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows a schematic representation of the key steps in nitrogenutilization in a plant cell. Nitrate (NO₃ ⁻) is transported into theplant cell and converted to nitrite (NO₂ ⁻) by nitrate reductase (NR).Nitrite is translocated from the cytoplasm to the chloroplast where itis reduced by nitrite reductase (NiR) to ammonium (NH₄ ⁺). Glutaminesynthetase (GS) functions in assimilating or recycling ammonium. Anenzyme couple, glutamine synthetase (GS)/glutamate synthase (GOGAT),catalyzes the conversion of glutamine (Gln) to glutamate (Glu).Glutamate is a building block of many amino acids. In addition, alanineis synthesized by the enzyme alanine aminotransferase (AlaAT) frompyruvate and glutamate in a reversible reaction.

FIG. 2 shows the nucleotide sequence for the OSAnt1 promoter of thepresent invention (SEQ ID NO:1). The sequence was isolated using ablastn search of the NCBI database using the nucleotide sequence(366-3175 bp) of the Brassica btg26 gene (Stroeher et al., 1995, PlantMol. Biol. 27:541-551) to identify the homologous rice nucleotidesequence (accession number AF323586). This sequence was then used inturn against the TIGR Oryza sativa sequencing project (see:tigr.org/tdb/e2k1/osa1/), as set out in Example 1. The putative TATA boxis shown in bold and the primers used in PCR amplifying the sequencefrom the rice genome are underlined.

FIG. 3 shows a schematic representation of the steps for producing thegenetic construct OsAnt1pro-Gus, using the reporter genebeta-glucuronidase (GUS) in accordance with the method described inExample 2.

FIG. 4 shows a schematic representation of the steps for producing thegenetic construct OsAnt1pro-AlaAT in accordance with the methoddescribed in Example 2.

FIG. 5 shows expression of the GUS reporter gene directed by the OsAnt1promoter of the present invention. Expression is present in the cellexpansion area of root tips of developing roots (panel A); in root hairsof developing roots (panel B); and in lateral roots of roots (panel C)of an Oryza sativa plant transformed with the genetic constructOsAnt1pro-Gus as shown in FIG. 3, in accordance with the methoddescribed in Example 3. Darkly stained areas indicate expression of theGUS reporter gene.

FIG. 6 shows the average dry weight biomass (grams) of Oryza sativaplants transformed with the genetic construct OsAnt1pro-AlaAT as shownin FIG. 4 compared to the average dry weight biomass (grams) of control,wild-type Oryza sativa plants grown under the same growth conditions asgiven in Example 3.

FIG. 7 shows the average total seed weight (grams) of seeds collectedfrom Oryza sativa plants transformed with the genetic constructOsAnt1pro-AlaAT as shown in FIG. 4 compared to the average total seedweight (grams) of seeds collected from control, wild-type Oryza sativaplants grown under the same growth conditions as given in Example 3.

FIG. 8 shows the relationship between dry weight biomass (grams) andtotal seed weight (grams) for each transgenic plant.

DETAILED DESCRIPTION

A promoter sequence obtained from rice, methods of using the promotersequence, and a rice plant and a portion of a rice plant including thepromoter sequence as described.

The following description is of a preferred embodiment.

A promoter sequence for directing expression of a coding region ofinterest within an Oryza sativa plant is described. The promotersequence is an isolated Oryza sativa antiquitin (OsAnt1) promotersequence including SEQ ID NO: 1 and active fragments thereof.

The language “coding region of interest” includes any gene that isdesirably expressed in one or more than one plant tissue. Examples of acoding region of interest which may advantageously be utilized inconjunction with the methods described herein include nucleic acidsequences that encode one or more than one protein involved in nitrogenassimilation, nitrogen utilization, or a combination thereof. Otherexamples would be nucleic acid sequences encoding one or more than oneprotein involved in nitrogen uptake and utilization.

By “promoter” it is meant the sequence of a DNA molecule that directstranscription of a downstream gene to which it is operatively linked orthat, when fused to a particular gene and introduced into a cell, causesexpression of the gene at a level higher than is possible in the absenceof the DNA sequence. Such promoters can be the full length promoter oractive fragments thereof. By “active fragment” is meant a fragment thathas at least about 0.1%, preferably at least about 10%, and morepreferably at least about 25% of the activity of a reference promotersequence as tested via methods known to those of skill in the art fordetecting promoter activity, e.g., measurement of GUS reporter genelevels. DNA sequences necessary for activity can be identified bysynthesizing various fragments and testing for expression or introducingpoint mutations in certain regions and testing for loss of activity.Heterologous fragments of promoters or other promoter sequences may becombined to mediate the activity of a promoter sequence. For example,the CaMV 35S promoter or other known promoter sequences may be combinedwith the promoter sequence described herein to mediate expression of acoding region of interest.

The gene constructs described herein can also include further enhancers,either translation or transcription enhancers, as may be required. Theseenhancer regions are well known to persons skilled in the art and caninclude the ATG initiation codon and adjacent sequences. The initiationcodon must be in phase with the reading frame of the coding sequence toensure translation of the entire sequence. The translation controlsignals and initiation codons can be from a variety of origins, bothnatural and synthetic. Translational initiation regions may be providedfrom the source of the transcriptional initiation region or from thestructural gene. The sequence can also be derived from the promoterselected to express the gene and can be specifically modified toincrease translation of the mRNA.

The gene constructs described herein can further include a 3′untranslated (or terminator) region that contains a polyadenylationsignal and other regulatory signals capable of effecting mRNA processingor gene expression. Nonlimiting examples of suitable 3′ regions are the3′ transcribed non-translated regions containing a polyadenylationsignal of Agrobacterium tumour-inducing (Ti) plasmid genes such as thenopaline synthase (Nos gene), plant genes such as the soybean storageprotein genes, and the small subunit of the ribulose-1,5-bisphosphatecarboxylase (ssRUBISCO) gene.

By “operatively linked” or “operative linkage” it is meant that theparticular sequences interact either directly or indirectly to carry outan intended function, such as mediation or modulation of geneexpression. The interaction of operatively linked sequences may bemediated, for example, by proteins that interact with the operativelylinked sequences.

The term “exogenous” as used herein in reference to a nucleic acidmolecule means a nucleic acid molecule originating from outside theplant. An exogenous nucleic acid molecule can have a naturally occurringor non-naturally occurring nucleotide sequence. One skilled in the artunderstands that an exogenous nucleic acid molecule can be aheterologous nucleic acid molecule derived from the same plant speciesor a different plant species than the plant into which the nucleic acidmolecule is introduced. Alternatively, it can be a nucleic acid moleculederived from a non-plant species such as fungi, yeast, bacteria or othernon-plant organisms.

Because the level of transgene expression in monocots is generallyhigher when driven by a monocot promoter rather than a dicot promoter,the rice genome was examined to identify a rice homologue of btg26. Thiswas done using a blastn search using the amino acid sequence of theBrassica btg26 gene (Stroeher et al., 1995, Plant Mol. Biol. 27:541-551)against the TIGR Oryza sativa sequencing project (see Example 1). TheOryza sativa antiquitin (OsAnt1) gene was identified on chromosome 9 ofrice and a promoter sequence, upstream (5′) to the ATG start codon ofOsAnt1 gene was identified (FIG. 2; SEQ ID NO: 1). This promotersequence was used to drive the expression of a coding region ofinterest. Plants expressing, for example but not limited to, AlaAT undercontrol of the OsAnt1 promoter sequence exhibited increased biomass,seed yield and NUE (See Example 3).

Therefore, the production of Oryza sativa plants that express one ormore target genes or coding regions of interest are described. Furtherprovided is a method for increasing biomass of a rice plant and a methodfor increasing seed yield of a rice plant. By the methods describedherein, it is possible to produce rice plants having one or more desiredtraits or properties; e.g., to alter specifically the genetic propertiesof the plant, the physiological properties of the plant, or both thegenetic and the physiological properties of the plant. In addition,methods of producing rice plants having expression in the roots of oneor more than one desired gene or coding region of interest, using theOsAnt1 promoter sequence disclosed herein are described.

The production of rice plants having expression of one or more than onetarget gene or coding region of interest is accomplished using theOsAnt1 promoter sequence of the present invention, which directsexpression of the coding region of interest. The OsAnt1 promotersequence enables expression of the target sequence in one or more thanone tissue of the rice plant, suitably in the root tissue.

It will be understood by one skilled in the art that modifications maybe made to the OsAnt1 promoter sequence useful in the methods andconstructs of the invention to improve or modulate the activity of thepromoter sequence. Selected regions of the OsAnt1 promoter sequence maybe operatively linked to a single target coding region to alter theexpression level of the linked coding region, or the OsAnt1 promotersequence may be operatively linked to one or more than one target codingregions such that the expression of each target coding region may becoordinately regulated. The OsAnt1 promoter sequence may be of any sizeappropriate to permit its functioning as a promoter. The OsAnt1 promotersequence may be modified using standard methods known within the art,for example but not limited to, mutagenesis, deletion, insertion,substitution, or truncation, to alter the degree to which theoperatively linked coding region is expressed, or to alter thespecificity of expression directed by the promoter sequence. Further,the placement of the OsAnt1 promoter sequence relative to theoperatively linked coding region may be modulated (e.g., moved furtheraway or closer together) to attain a desired level of promoter-directedexpression.

It is envisaged that the OsAnt1 promoter sequence may direct expressionof the coding region of interest in response to a specific environmentalor physiological condition. For example, the promoter sequence may beactivated under conditions of drought stress, osmotic stress, saltstress, temperature stress, nutrient deprivation, or under specificdevelopmental conditions for example but not limited to, upongermination, fruiting, or seed production.

A coding region of interest, or a target gene, of the invention may beany nucleotide sequence that is desirably expressed within a rice plant.General classes of coding regions which may be advantageously employedin the methods and constructs of the invention include nucleotidesequences encoding structural proteins; proteins involved in thetransport of nitrogen; proteins involved in the uptake of nitrogen;proteins involved in both the transport and uptake of nitrogen; enzymesand proteins involved in nitrogen utilization; proteins involved inplant resistance to pesticides or herbicides; proteins involved in plantresistance to nematodes, viruses, insects, or bacteria; proteinsinvolved in plant resistance to stress, for example but not limited toosmotic, temperature, pH, or oxygen stress; proteins involved instimulation or continuation of plant growth; proteins involved inphytoremediation; or proteins having pharmaceutical properties orencoding enzymes which produce compounds having pharmaceuticalproperties.

For example, the coding region of interest may encode a nitrogenutilization protein and, in particular, an enzyme that assimilatesammonia into amino acids or uses the formed amino acids in biosyntheticreactions. This protein may be selected from, but not limited to, anitrate transporter (high or low affinity), an ammonium transporter, anammonia transporter, an amino acid transporter, alanine dehydrogenase,glutamine synthetase (GS), asparagine synthetase (AS), glutamatesynthase (also known as glutamate 2:oxogluturate amino transferase andGOGAT), asparaginase (ANS), glutamate dehydrogenase (GDH), nitratereductase, aspartate aminotransferase (AspAT), alanine aminotransferase(AlaAT), and other known aminotransferases. Such proteins are disclosedin US Patent Application Publication Number 2005/0044585, which ishereby incorporated by reference in its entirety.

The target gene or coding region of interest may be naturally expressedin the rice plant or it may be heterologous to the rice plant. The genemay originate from any source, including viral, bacterial, plant oranimal sources. Preferably, the coding region of interest isheterologous to the OsAnt1 promoter sequence to which it is operativelylinked, in that it is not from the gene the OsAnt1 promoter sequence isnaturally linked to.

The coding region can be modified in any suitable way in order toengineer a gene or rice plant with desirable properties. The codingregion can be modified to be transcribable and translatable in the plantsystem; for example, the nucleotide sequence encoding the protein ofinterest can be modified such that it contains all of the necessarypoly-adenylation sequences, start sites and termination sites whichallow the coding sequence to be transcribed to mRNA (messengerribonucleic acid) and the mRNA to be translated in the rice plant.Further, the coding region may be modified such that its codon usage ismore similar to that of native genes of the rice plant (i.e., plantoptimized sequence may be used). Such nucleotide sequence modificationsand the methods by which they may be made are well known to one of skillin the art.

The constructs described herein include an OsAnt1 promotersequence-coding region of interest are most efficiently introduced intoa rice plant, a rice plant cell or plant protoplast through the use of avector. Examples of cloning or expression vectors suitable for use withthe invention are plasmids (such as pAG001), cosmids, viral DNA or RNA,and minichromosomes. Appropriate plant vectors are well known in the art(see, e.g., Clark, M., ed. (1997) Plant Molecular Biology: A LaboratoryManual. Springer Verlag, ISBN: 3540584056, hereby incorporated byreference in its entirety).

Vectors can advantageously contain one or more bacterial orplant-expressible selectable or screenable markers or reporter genes,such that incorporation of the vector into a rice plant cell orprotoplast can be monitored. It is preferable that such selectable orscreenable markers confer a readily detectable phenotype, such asresistance to an otherwise toxic compound (e.g., kanamycin resistance)or a calorimetric or luminescent reaction upon incubation of the plantwith an appropriate substrate (e.g., beta-glucuronidase (GUS) orluciferase genes). Such reporter genes are well known in the art.

The constructs described herein can be introduced to a rice plant orplant cell by any useful method. A large number of processes areavailable and are well known to deliver genes to plant cells. One of thebest-known methods involves the use of Agrobacterium or similar soilbacteria as a vector, wherein the Agrobacterium is transformed with theconstruct of interest or a vector containing the construct. Targettissues of a plant are co-cultivated with the transformed Agrobacteriumwhich inserts the nucleotide sequence of interest into the plant genome,as is described by U.S. Pat. No. 4,940,838 (Schilperoort et al.; whichis incorporated herein by reference in its entirety), and Horsch et al.(1985, Science 227:1229-1231; which is incorporated herein byreference). Alternative gene transfer and transformation methods usefulin the present invention include, but are not limited to, liposomes,electroporation or chemical-mediated uptake of free DNA, calciumphosphate co-precipitation techniques, targeted microprojectiles andmicro- or macroinjection, direct DNA transformation, and may involve Tiplasmids, Ri plasmids, or plant virus vectors. Such transformationmethods are well documented in the art. It will be understood by oneskilled in the art that the method chosen for rice plant, rice plantcell, or protoplast transformation will in large part be determined bythe nature of the OsAnt1 promoter sequence-target sequence construct, orthe vector containing the construct.

A transformed Oryza sativa plant including a coding region in operativelinkage with an OsAnt1 promoter sequence is described. Also provided isOryza sativa plant seed including a coding region in operative linkagewith the OsAnt1 promoter sequence described herein. The transformed riceplants and seeds produced according to the present invention may befurther useful in breeding programs for the production of rice plantshaving more than one desired trait. For example, two transformed riceplants of the invention each having expression of a desired transgenemay be crossed using known methods to produce progeny that arecharacterized in having expression of both transgenes. In this manner,it is possible to produce transformed rice plants having a combinationof desirable traits expressed in the plant.

Furthermore, it will be understood by one skilled in the art thatdifferent varieties of plants may be more or less amenable to geneticmanipulation in general; therefore, it may be advantageous to firsttransform a related varieties of the rice plant by the methods and withthe constructs described herein and to subsequently introduce expressionof the target gene into the rice plant by cross-breeding techniques.Such techniques and appropriately related plant varieties are well knownto one skilled in the art.

Seeds may be harvested from transformed rice plants using methods wellknown in the art and further used to re-grow the transformed plants andhybrids described herein.

The methods and constructs described herein allow the production of riceplants and seeds having expression of one or more desired genes in therice plant. There is a wide variety of possible applications of theplants described herein, including, but not limited to, the productionof rice plants having increased stress tolerance, improved nitrogenuptake, improved nitrogen utilization, improved nutrient content,improved nutrient yields of desired compounds, and phytoremediativeproperties. Specific applications are further described below.

The plants described herein are able to thrive on nutrient-poor soils.It is well known in the art that certain plant species, particularlycrop plants, deplete the soil of nutrients necessary to sustain growth,such as nitrogen, phosphate, and potassium. In order to replenish thelacking nutrients, it is necessary either to fertilize the soil (anexpensive and environmentally damaging procedure) or to cultivate plantsknown to deposit the depleted nutrient into the soil (e.g., clover orsoybean in the case of nitrogen depletion). However, these alternativesmay be less economically acceptable. The methods described herein permitthe targeted expression of nucleotide sequences involved in nutrientuptake (for example, transport molecules) to those tissues in which theuptake occurs (for example, the root or root hairs) to thereby improvethe ability of the rice plant to absorb the nutrients from theenvironment.

The methods described herein may also be used to produce rice plantsthat express heterologous or optimized native nutrient utilization genesor coding regions that permit more efficient use of the nutrient, suchthat less of the nutrient (for example, nitrogen) is required for thenormal growth and functioning of the plant. Further, it is possible,using the methods described herein, to express coding regions ofinterest involved in the use and uptake of nutrients not normally usedby the rice plant suitable in those plant tissues that are directlyexposed to the different nutrient (for example, root and leaf). In thismanner, rice plants that are able to grow and thrive on differentnutrient sources (for example, different nitrogen sources) may beproduced. Particularly useful target genes for the optimization ofnitrogen efficiency of the plant include: a nitrate transporter (high orlow affinity), an ammonium transporter, an ammonia transporter, an aminoacid transporter, alanine dehydrogenase, glutamine synthetase (GS),asparagine synthetase (AS), glutamate synthase (also known as glutamate2:oxogluturate amino transferase and GOGAT), asparaginase (ANS),glutamate dehydrogenase (GDH), nitrate reductase, and anaminotransferase such as alanine aminotransferase (AlaAT) or aspartateaminotransferase (AspAT) such as those described in US PatentApplication Publication Number 2005/0044585, which is herebyincorporated by reference in its entirety.

The techniques described herein may be used to produce rice plants thatcan more efficiently utilize fertilizer input by rapidly taking up thenitrogen provided by the fertilizer and storing it at the time ofapplication, thereby reducing the amounts of nitrogenous fertilizer lostto leaching, etc. This may permit a reduction in the amount ofnitrogenous fertilizer required to be applied to a rice crop to obtaincrop yields comparable to those obtained using normal cultivationtechniques and rice plants that have not been modified according to thepresent invention. Additional agronomic advantages can include fastergrowth and rice crop yield, where nitrogenous fertilizer input ismaintained at levels used in common crop cultivation techniques.

Transformed Oryza sativa plants expressing an oligonucleotide sequenceencoding alanine aminotransferase (AlaAT) in operative linkage with theOsAnt1 promoter sequence of the present invention were produced. Asshown in Example 3, transformed plants expressing AlaAT under control ofthe OsAnt1 promoter sequence exhibited higher dry weight biomass andincreased seed yields when compared to control plants. These resultsindicate that rice plants expressing a heterologous AlaAT under thecontrol of OsAnt1 promoter sequence are capable of optimizing theutilization of available nitrogen thereby resulting in an increase inplant biomass, seed yield or a combination thereof.

Therefore, methods for increasing biomass of an Oryza sativa plant,including providing an Oryza sativa plant with a coding region ofinterest in operative linkage with an OsAnt1 promoter sequence describedherein are described. The coding region of interest may encode an enzymeinvolved in optimizing nitrogen efficiency of the plant, for example, itmay encode alanine aminotransferase (AlaAT) and expression of the codingregion suitably takes place in the root.

Also provided is a method for increasing seed yield of an Oryza sativaplant, including providing an Oryza sativa plant with a coding region inoperative linkage with an OsAnt1 promoter sequence described in thepresent invention. The coding region of interest may encode an enzymeinvolved in optimizing nitrogen efficiency of the plant, for example, itmay encode alanine aminotransferase (AlaAT).

The above description is not intended to limit the claimed invention inany manner; furthermore, the discussed combination of features might notbe absolutely necessary for the inventive solution.

The following examples further illustrate certain embodiments of theinvention.

EXAMPLES Example 1 Isolation and Characterization of the OsAnt1 PromoterSequence

The nucleotide sequence (bp 366-3175) of the btg26 gene (Stroeher etal., 1995, Plant Mol. Biol. 27:541-551; accession number S77096) wasused to search the nucleotide database at NCBI using the blastn searchtool. A rice sequence (accession number AF323586) was identified andthis nucleotide sequence was used to search the TIGR Oryza sativasequencing project (tigr.org/tdb/e2k1/osa1/). The rice homologue ofbtg26, Oryza sativa antiquitin (OsAnt1), was identified on chromosome 9of rice (accession number AP005570; 98524-101189 base pairs). A 973-bpsequence upstream of the start codon of OsAnt1 is shown in FIG. 2 (SEQID NO:1). The sequence of the 403 bps upstream (5′) of the ATG startcodon of the OsAnt1 gene was selected for analysis. To determine if thesequence was likely to function as a promoter sequence, the sequence wasanalyzed using the TSSP plant promoter prediction software found athttp://softberry.com/. The analysis predicted that the sequence was aplant promoter sequence. The most likely location of the TATA box (boldin FIG. 2), as well as other promoter sequence elements, was determined.

Since the projected OsAnt1 promoter sequence was predicted to containpromoter elements according to the Softberry analysis, the sequenceswere analyzed for promoter motifs that may be recognition sites fortranscription factors using Signal Scan Software (Prestridge, 1991;http://bimas.dcrt.nih.gov/molbio/signal). Five different signalsequences were predicted in the OsAnt1 promoter, including ADR1, DBF-A,GAL4, HSTF and RAF transcription factor binding sites.

The OsAnt1 sequence was compared to nucleic acid sequences of btg26promoter sequences from Brassica napus and Arabidopsis using theClustalW 1.8 multiple sequence alignment software on the BCM SearchLauncher homepage (searchlauncher.bcm.tmc.edu/) and BOXSHADE server(ch.embnet.org/software/BOX_form.html). Inspection of conservednucleotides revealed that the Brassica and Arabidopsis turgor gene-26promoter sequences are more similar to each other than to the OsAnt1sequence. A feature among all three promoter sequences is thepolypyrimidine (CT) tracts evident within the nucleotide sequences.These tracts range from 20-22 bases and are found just upstream of theprobable TATA boxes in all three promoter sequences. Furthermore, theOsAnt1 sequence has a second polypyrimidine tract just upstream of theATG start codon.

Rice genomic DNA was isolated from cv. Kitaake. The following PCRprimers (positions underlined in FIG. 2) corresponding to the OsAnt1promoter region were selected:

Primer 1: AGGAAGTGATTTTTAGCGTAGCTG; (SEQ ID NO: 2) Primer 2:ATGGCAGAAGAGAGAGAGAGAGAGG. (SEQ ID NO: 3)Touch-down PCR was conducted using rice genomic DNA and the aboveprimers. A 975-bp fragment was produced. The amplified PCR fragment wasligated into pCR®II-TOPO vector (Invitrogen) and transformed into E.coli, TOP 10 cells. The resulting plasmid is designatedpT-riceOsAnt1pro.

Sequence analysis indicated that the 975-bp PCR fragment encodes apromoter sequence designated the OsAnt1 promoter sequence. Comparison ofthe OsAnt1 promoter from cv. Kitaake with that of cv. Nipponbare(obtained from the database) revealed that they share 99.9% identity.The putative TATA box was found 145 bps upstream of the start codon.

Example 2 Genetic Constructs Containing OsAnt1 Promoter Sequence

Genetic constructs containing OsAnt1 promoter sequence driving thebeta-glucuronidase (GUS) reporter gene (OsAnt1pro-Gus) or the barleyAlaAT gene (OsAnt1pro-AlaAT) were produced using the steps shownschematically in FIGS. 3 and 4, respectively.

RiceOsAnt1pro-GUS Construct

The RiceOsAnt1pro-GUS construct was produced by amplifying thepT-RiceOsAnt1pro template using the following primers:

Primer 3: EcoRI-OsAnt1 promoter sequence GGAATTCAGGAAGTGATTTTT (SEQ IDNO: 4) Primer 4: NcoI-OsAnt1 promoter sequence CATGCCATGGATGGCAGAAGA(SEQ ID NO: 5)The resultant PCR fragments were ligated into the plant binary vector,pCAMBIA1305.1, digested with EcoR1 and Nco1 to produce apCAMBIA1305.1-riceOsAnt1pro-GUS construct. The EcoRI and NcoI sequencesat the end of primers 3 and 4, respectively, allowed insertion of thePCR fragment into the pCAMBIA1305.1 vector, replacing the existingCaMV35s promoter with the OsAnt1 promoter sequence. The NcoI sequence(CCATGG) includes a Met codon, ATG, which is in frame with the GUSreporter gene and allows expression of the GUS reporter gene from theOsAnt1 promoter sequence.RiceOsAnt1pro-AlaAT Construct

The RiceOsAnt1pro-AlaAT construct was produced by amplifying thepT-RiceOsAnt1pro template using the following primers:

Primer 3: EcoRI-OsAnt1 promoter sequence GGAATTCAGGAAGTGATTTTT (SEQ IDNO: 4) Primer 5: PstI-OsAnt1 promoter sequence AACTGCAGATGGCAGAAGA (SEQID NO: 6)and the resultant PCR fragments digested with EcoR1 and Pst1 wereligated into the plant binary vector, pCAMBIA1300, digested with EcoR1and Pst1 to produce pCAMBIA1300-riceOsAnt1pro.

An AlaAT DNA fragment was amplified by PCR using pAG001 as a template.pAG001 is described in U.S. Pat. No. 6,084,153 where it is identified aspbtg26/AlaAT/nos. It contains the btg26 promoter linked to the barleyAlaAT gene with a nopaline synthase terminator. The barley AlaAT/nosterminator sequences were amplified from pAG001 using the followingprimers:

Primer 6: PstIA1aAT sequence AACTGCAGATGGCTGCCACCG (SEQ ID NO: 7) Primer7: HindIII-NOS terminator sequence CCCAAGCTTCCCGATCTAGTA (SEQ ID NO: 8)

The resulting AlaAT/nos fragment was digested with Pst and HindIII andligated into the pCAMBIA1300-riceOsAnt1pro digested with Pst1 andHindIII to produce a pCAMBIA1300-riceOsAnt1pro-AlaAT construct.

Example 3 Transformation of Rice Plants with Vectors Including OsAnt1

pCAMBIA1305.1-riceOsAnt1pro-GUS and pCAMBIA1300-riceOsAnt1pro-AlaAT weretransferred into Agrobacterium strain EHA105 (Hood et al., (1993)Transgenic Res. 2: 208-218) by electroporation (Sambrook et al. 1989 inMolecular Cloning, A Laboratory Manual Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press). Agrobacterium cells were plated onsolid AB medium (Chilton et al. 1974) containing 50 mg/l kanamycin andincubated at 28° C. for 3 days. The bacteria were then collected with aflat spatula and resuspended in liquid co-cultivation medium (R2-CL,Table 1) by gentle vortexing prior to transforming the rice tissues.

Transformation of Rice

Mature seeds of rice (Oryza sativa L. cv. Nipponbare) were used in thetransformation experiment. The seeds were dehusked and surfacesterilized with 50% bleach plus 0.1% Tween-20 for 10 min followed bydipping (1 min) in 70% (v/v) ethanol and then rinsing five times insterile distilled water. Following sterilization, seeds were cultured oncallus induction medium (NB, Table 1) and incubated for three weeks inthe dark at 28° C.

TABLE 1 Medium used for callus induction, inoculation, co-culture,resting phase, selection, regeneration and rooting Medium CompositionNB^(a) N6 major salt and iron source (Chu (1975) Sci. Sin. 5: 659-Callus induction medium 668) + B5 major salts and vitamins (Gamborg etal. (1968) (filter sterilize) Exp. Cell Res. 50: 151-158) + 3AA (100mg/l L-tryptophan + 500 mg/l L-proline + 500 mg/l L-glutamine) + 500mg/l casein hydrolysate + 2.0 mg/1 2,4-D + 0.5 mg/1 picloram + 30 g/1sucrose, pH 5.8, 0.3% gelrite R2-CL R2 major and minor salts, vitaminsand iron source without Liquid co-culture medium sucrose (Ohira et al.(1973) Plant and Cell Physiol. (filter sterilize) 14: 1113-1121) + 0.25M glucose + 125 μM acetosyringone + 10 mM MES buffer, pH 5.2 + 50 mMpotassium phosphate buffer, pH 5.2 + 400 mg/l L-cysteine + 2.0 mg/12,4-D + 0.5 mg/l picloram + 0.5 mg/l BAP, pH 5.2 R2-CS R2 major andminor salts, vitamins and iron source without Solid co-culture mediumsucrose (Ohira et al. (1973) Plant and Cell Physiol. (filter sterilize)14: 1113-1121) + 0.25 M glucose + 125 μM acetosyringone + 10 mM MESbuffer, pH 5.2 + 50 mM potassium phosphate buffer, pH 5.2 + 400 mg/lL-cysteine + 2.0 mg/1 2,4-D + 0.5 mg/l picloram + 0.5 mg/l BAP, pH 5.2 +0.3% gelrite R2-AS R2 major and minor salts, vitamins and iron sourcewithout Resting phase sucrose + 0.25 M sucrose + 0.5 mM acetosyringone +10 (filter sterilize) mM MES buffer, pH 5.0 + 50 mM potassium phosphatebuffer, pH 5.0 + 10 mM CaCl₂ + 400 mg/l L-cysteine + 2.0 mg/1 2,4-D +0.5 mg/l picloram + 0.5 mg/l BAP + 250 mg/l cefotaxime + 250 mg/lamoxicillin, pH 5.0, 0.3% gelrite R2S R2 major and minor salts, vitaminsand iron source + 30 g/l Selection medium sucrose + 2.0 mg/1 2,4-D + 0.5mg/l picloram + 50 mg/l (filter sterilize) hygromycin + 250 mg/lcefotaxime + 100 mg/l amoxicillin, pH 5.8, 0.3% gelrite NBS NB medium +3AA + 2.0 mg/1 2,4-D + 0.5 mg/l Picloram + Selection medium-II 50 mg/lhygromycin + 250 mg/l cefotaxime + 100 mg/l (filter sterilize)amoxicillin, pH 5.8, 0.3% gelrite PRN NB medium + 3AA + 5 mg/l ABA + 2mg/l BAP + 0.5 Pre-regeneration medium mg/l NAA + 50 mg/l hygromycin +100 mg/l cefotaxime + (filter sterilize) 50 mg/1 amoxicillin, pH 5.8,0.4% gelrite RN NB medium + 3 mg/1 BAP + 0.5 mg/l NAA + 50 mg/lRegeneration medium hygromycin + 100 mg/l cefotaxime + 50 mg/lamoxicillin, (filter sterilize) pH 5.8, 0.4% gelrite R ½MS (Murashigeand Skoog (1962) Physiol Plant 15: 473- Rooting medium 497) + 50 mg/lhygromycin^(b) + 100 mg/l cefotaxime + 50 (Autoclave/filter sterilize)mg/l amoxicillin, pH 5.8, 0.3% gelrite ^(a)NB medium with 1.25 mg/lCUSO₄ ^(b)Optional

After three weeks, 3-5 mm long embryogenic nodular units released fromthe scutellum-derived callus at the explant/medium interface wereimmersed into 25 ml of liquid co-culture medium (R2-CL, Table 1)containing Agrobacterium cells at the density of 3-5×10⁹ cells/ml(OD₆₀₀=1) in a 100 mm-diameter Petri dish for 10-15 minutes. Embryogenicunits were then blotted dry on sterilized filter paper, transferred to aPetri dish containing solid co-culture medium (R2-CS, Table 1) andincubated for three days at 25° C. in the dark. Co-cultured embryogeniccalli were then transferred to resting medium (R2-AS, Table 1) andincubated at 28° C. in the dark for a week.

After a week, uncontaminated embryogenic units were then individuallytransferred to selection medium (R2S, Table 1) containing hygromycin forselection of transformed tissue and incubated at 28° C. in the dark.Following 3 weeks of selection on R2S medium, the embryogenic units thatturned dark brown with brownish protuberances arising throughout thecallus surface were transferred to NBS selection medium (Table 1). After5 weeks of co-culture, the protuberances developed into brownishglobular structures that were gently teased apart from callus andincubated for 2 weeks in the resealed Petri dish. After 2 weeks, theseglobular structures converted into round shaped, compact and yellowishcalli.

The putatively transgenic, hygromycin-resistant calli were gently pickedout, transferred, cultured on pre-regeneration medium (PRN, Table 1) andthen incubated for a further week. All of the resistant callioriginating from a single co-cultured embryogenic nodular unit weregrouped in a sector of the PRN dish. Creamy-white, lobed calli with asmooth and dry appearance were individually transferred to regenerationmedium (RN, Table 1), incubated for 2 days in the dark, then maintainedfor three weeks under a 12/12-h (day/night) photoperiod with lightprovided at an intensity of 55 μmol/m per sec. Green shoots regeneratingfrom a resistant callus were dissected and sub-cultured in test tubecontaining rooting medium (R, Table 1) for 1-2 weeks to promote vigorousroots and tillers before being transferred to pots in the growth rooms.Transgenic plants were grown to maturity in 16-cm pots containingsoil-less potting mixture (Metromix 220). Plants were maintained ingrowth rooms set to 28° C. and 14/10 hours day/night photoperiods.Fertilizer was applied twice a week starting two weeks after planting inpots. The fertilizer mix contained 225 g 20/20/20 fertilizer, 50 g ofplant micronutrients, 6.1 g of CuSO₄.5H₂O, 140 g FeEDTA, 13.8 gZnSO₄.7H₂0, 260 g MgSO₄.7H₂O, 3.7 g H₃BO₃ for a total of 712.4 g. Twograms of the fertilizer mix are dissolved in 8 liters of water andapplied twice a week to 24 plants.

Analysis of Expression Directed by the OsAnt1Promoter Sequence

Induction of expression directed by the OsAnt1 promoter sequence wasexamined using rice plants transformed with the OsAnt1pro-GUS construct.Plants were germinated and grown hydroponically in sterile conditions inMagenta jars. Two-week-old plants were stained for in vivo GUS activityby injecting into the root media 5 mls of 50 mM phosphate buffer (pH7.5) containing 0.2 mM X-gluc(5-bromo-4-chloro-3-indolyl-beta-glucuronic acid) and incubating theplants in this media for 1-24 hours. Root tissue was then viewed under adissection microscope and photographs were taken, which are shown inFIG. 5.

Dark stained areas in FIG. 5 indicate expression of the GUS reportergene. There is no expression of the GUS reporter gene driven by theOsAnt1 promoter in the root tip (specifically the dividing cells);however, expression begins very quickly in the cell expansion zone, justbehind the root tip. The OsAnt1 promoter sequence directed expression ofthe GUS reporter gene in the root hairs as well. Further from the roottip in more mature roots, expression is lost from the main root, butlateral roots stain very heavily, indicating that OsAnt1 directsexpression in these lateral roots very strongly.

Analysis of Transformed Plants Containing the AlaAt Construct

Fifty-eight OsAnt1/AlaAT/NOS transgenic plants were generated andmeasurements for flowering, tiller number, seed weights and biomass atmaturity were recorded for the T₀ generation plants.

The dry weight biomass of OsAnt1/AlaAT plants and control plants wasmeasured at maturity, and the data is presented in FIG. 6. The averagebiomass of the transgenic OsAnt1/AlaAT plants was higher than theaverage biomass of control plants.

Seeds were collected from OsAnt1/AlaAT plants and control plants atmaturity and the total weight of the seeds was measured. The results areshown in FIG. 7, which shows that the total seed weight of seedscollected from OsAnt1/AlaAT plants was higher than that of the seedweight from control plants.

FIG. 8 shows the relationship between dry weight biomass and total seedweight for each transgenic plant. A substantially linear correlation isshown, which indicates that an increase in biomass results in acorresponding increase in total seed weight in OsAnt1/AlaAT plants.

These results indicate that OsAnt1/AlaAT transgenic plants are capableof optimizing the utilization of available nutrients thereby resultingin an increase in plant biomass, seed yield or a combination thereof.

All citations listed herein are hereby incorporated by reference intheir entirety.

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined in the claims.

1. A method for directing expression of a target gene in an Oryza sativa plant comprising: contacting an Oryza sativa plant with a target gene in operative linkage with an OsAnt1 promoter sequence comprising SEQ ID NO: 1 or an active fragment thereof; and introducing into the plant the target gene in operative linkage with an OsAnt1 promoter sequence comprising SEQ ID NO: 1 or an active fragment thereof, wherein the OsAnt1 promoter directs expression of the target gene in the plant.
 2. The method of claim 1, wherein the target gene encodes a nitrogen utilization protein selected from the group consisting of a high affinity nitrate transporter, a low affinity nitrate transporter, an ammonium transporter, an ammonia transporter, an amino acid transporter, alanine dehydrogenase, glutamine synthetase, asparagine synthetase, glutamate synthase, glutamate 2:oxogluturate amino transferase, asparaginase, glutamate dehydrogenase, nitrate reductase, aspartate aminotransferase and alanine aminotransferase.
 3. The method of claim 2, wherein the nitrogen utilization protein is alanine aminotransferase.
 4. The method of claim 1, wherein the plant is transformed with the genetic construct of claim
 5. 5. The method of claim 1, wherein expression of the target gene takes place in the root of the plant.
 6. A method for increasing biomass of an Oryza sativa plant, comprising: contacting an Oryza sativa plant with a target gene in operative linkage with an OsAnt1 promoter sequence comprising SEQ ID NO: 1 or an active fragment thereof; and introducing into the plant the target gene in operative linkage with an OsAnt1 promoter sequence comprising SEQ ID NO: 1 or an active fragment thereof; wherein the OsAnt1 promoter directs expression of the target gene to obtain an Oryza sativa plant with increased biomass.
 7. The method of claim 6, wherein the target gene encodes a nitrogen utilization protein selected from the group consisting of: a high affinity nitrate transporter, a low affinity nitrate transporter, an ammonium transporter, an ammonia transporter, an amino acid transporter, alanine dehydrogenase, glutamine synthetase, asparagine synthetase, glutamate synthase, glutamate 2:oxogluturate amino transferase, asparaginase, glutamate dehydrogenase, nitrate reductase, aspartate aminotransferase and alanine aminotransferase.
 8. The method of claim 7, wherein the nitrogen utilization protein is alanine aminotransferase.
 9. The method of claim 6, wherein the plant is transformed with the genetic construct of claim
 5. 10. The method of claim 6, wherein expression of the target gene takes place in the root of the plant.
 11. A method for increasing seed yield of an Oryza sativa plant, comprising: contacting an Oryza sativa plant with a target gene in operative linkage with an OsAnt1 promoter sequence comprising SEQ ID NO: 1 or an active fragment thereof; and introducing into the plant the target gene in operative linkage with an OsAnt1 promoter sequence comprising SEQ ID NO: 1 or an active fragment thereof, wherein the OsAnt1 promoter directs expression of the target gene to obtain an Oryza sativa plant with increased seed yield.
 12. The method of claim 11, wherein the target gene encodes a nitrogen utilization protein selected from the group consisting of a high affinity nitrate transporter, a low affinity nitrate transporter, an ammonium transporter, an ammonia transporter, an amino acid transporter, alanine dehydrogenase, glutamine synthetase, asparagine synthetase, glutamate synthase, glutamate 2:oxogluturate amino transferase, asparaginase, glutamate dehydrogenase, nitrate reductase, aspartate aminotransferase and alanine aminotransferase.
 13. The method of claim 12, wherein the nitrogen utilization protein is alanine aminotransferase.
 14. The method of claim 11, wherein the plant is transformed with the genetic construct of claim
 5. 15. The method of claim 11, wherein expression of the target gene takes place in the root of the plant. 